2-Substituted (N)-Methanocarba A₃ Adenosine Receptor Agonists: In Silico, In Vitro, and In Vivo Characterization

Abstract

2-Arylethynyl (N)-methanocarba adenosine 5′methylamides are selective A 3 adenosine receptor (AR) agonists containing a preestablished receptor-preferred pseudoribose conformation. Here, we compare analogues having bulky 2substitution, either containing or lacking an ethynyl spacer between adenine and a cyclic group. 2-Aryl compounds 9−11, 13, 14, 19, 22, 23, 27, 29, 31, and 34, lacking a spacer, had human (h) A 3 AR K i values of 2−30 nM, and others displayed lower affinity. Mouse (m) A 3 AR affinity varied, with 2-arylethynyl having a higher affinity than 2-aryl analogues (7, 8 > 3c, 3d > 3b). However, 2-aryl-4′truncated derivatives had greatly reduced hA 3 AR affinity, even containing affinity-enhancing N 6 -dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical A 3 AR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hA 3 AR miniGα i recruitment assay, 31 (MRS8062) was (slightly) more potent compared to a β-arrestin2 recruitment assay, both in engineered HEK293T cells, and its maximal efficacy (E max ) was much higher (165%) than reference agonist NECA's. Thus, in the 2aryl series, A 3 AR affinity and selectivity were variable and generally reduced compared to the 2-arylethynyl series, with a greater dependence on the specific aryl group present. Selected compounds were studied in vivo in an ischemic model of peripheral artery disease (PAD). Rigidified 2-arylethynyl analogues 3a−3c were protective in this model of skeletal muscle ischemia-reperfusion injury/claudication, as previously shown only for moderately A 3 AR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the A 3 AR agonist SAR for (N)-methanocarba adenosines.

Lipid Trolling to Optimize A3 Adenosine Receptor-Positive Allosteric Modulators (PAMs)

“…We used the same de novo model we recently introduced, which contains a bound adenosine as an agonist and the α subunit of the G i protein . This model was compared to the recently released hA 3 AR structures (coordinates released while this manuscript was under review), and the structure and our model were shown to have an RMSD of ∼1.4 Å, which is lower than the structural resolution . Since 3 and 27 are structurally similar, we performed template-docking with the Glide-SP protocol, starting from the validated pose of 3 to obtain a putative 27 binding conformation within the allosteric pocket.…”